1. Field of the Invention
The present invention relates to the packaging of material for delivery, for example in gene therapy and other situations.
2. Description of Related Art
Gene transfer, as a means to effect gene therapy, is becoming increasingly important. Numerous studies have been carried out that seek to harness the inherent ability of viruses to infect eukaryotic cells in order to introduce a selected gene into a suitable recipient host (as reviewed: Anderson, 1992; Morgan and Anderson, 1993; Mulligan, 1993). Adenoviruses have proved particularly attractive for this purpose in that their genome can be manipulated to incorporate up to about 8 kbp of foreign DNA and, unlike retroviral vectors, transduced genes, can be expressed in non-dividing cells. One particular handicap shared by all the present virus vectors, however, is that they introduce unwanted viral genetic information alongside the gene of interest into the recipient host. The disadvantages of this unwanted viral material remain to be determined fully, but there is a great need for means to carry out improved genetic transfer without introduction of unwanted viral genes into target cells.
Physical, non-viral gene transfer methods such as chemical and mechanical techniques and membrane fusion-mediated transfer via liposomes have been used (Morgan and Anderson, 1993). However, the efficiency of transfer, in terms of expression of the genetic material transferred, is low. In other words, these methods are inefficient at transferring genetic material into cells in a stable manner so that the material is biologically functional.
The empty capsids of papovaviruses such as the mouse polyoma virus have received attention as possible vectors for gene transfer.
Barr et al, 1979, first described the use of polyoma empty particles when polyoma DNA and purified empty capsids were incubated in a cell-free system. The DNA of the new particle was protected from the action of pancreatic DNase. Slilaty and Aposhian, 1983, describe the use of those reconstituted particles for transferring a transforming polyoma DNA fragment to rat FIII cells. The empty capsids and reconstituted particles consist of all three of the polyoma capsid antigens VP1, VP2 and VP3 and there is no suggestion that pseudocapsids consisting of only the major capsid antigen VP1, could be used in genetic transfer.
Montross et al. 1991, described only the major capsid antigen, the cloning of the polyoma virus VP1 gene and its expression in insect cells. Self-assembly of empty pseudocapsids consisting of VP1 is disclosed, and pseudocapsids are said not to contain DNA. It is also reported that DNA inhibits the in vitro assembly of VP1 into empty pseudocapsids, which suggests that said pseudocapsids could not be used to package exogenous DNA for transfer to host cells. The results of Sandig et al. 1993, showed that empty capsids incorporating exogenous DNA could transfer DNA in a biologically functional manner to host cells only if the particles consisted of all three polyoma capsid antigens VP1, VP2 and VP3. Pseudocapsids consisting of VP1 were said to be unable to transfer the exogenous DNA so that it could be expressed in the host cells. We now know that this was probably because Ca2+ ions were not included in the medium in which the pseudocapsids were prepared. Haynes et al (1993) J. Virol., 67, 2486-2495) discuss the effect of calcium ions on empty VP1 pseudocapsid assembly. Hagensee et al (1993) J. Virol., 67, 315-322 disclosed the production of L1 and L1/L2 human papillomavirus capsids, but did not suggest their use to carry exogenous material.
The present invention aims to overcome the above problems associated with known viral and non-viral methods of exogenous material transfer by providing a method involving the use of a pseudocapsid consisting of a papovavirus major capsid antigen to transfer exogenous material to a host cell wherein that material is biologically functional.
The invention also aims to provide a pseudocapsid suitable for use in the above transfer methods, and a method of making a pseudocapsid.
The invention also aims to provide a pharmaceutical composition comprising the pseudocapsid of the invention and a pharmacologically acceptable carrier.
According to the invention there is provided a method of transferring material into a host cell comprising providing a pseudocapsid formed from papovavirus major capsid antigen and excluding minor capsid antigens, which pseudocapsid has exogenous material associated therewith; and treating the host cell with the pseudocapsid so that the material is taken up by the cell and is biologically functional in that cell.
By xe2x80x9cpseudocapsidxe2x80x9d we mean a structure in which the only papovavirus capsid antigen is the major capsid antigen without minor capsid antigens, which structure is hollow so that it can contain exogenous material.
The term xe2x80x9cpapovavirusxe2x80x9d defines a general family of viruses including polyoma virus (a mouse, virus), simian virus 40 (SV40), human variants (such as BK and JC) and papillomaviruses including human and bovine variants and other members. In each case, there is a major capsid antigen and one or more minor capsid antigens. For example, in papillomavirus the major antigen is L1 and the minor antigen is L2. In the present invention, the xe2x80x9cpseudocapsidsxe2x80x9d are formed from the major capsid antigen and not the minor antigen(s).
The term xe2x80x9cmajor capsid antigenxe2x80x9d includes conservative variants thereof capable of assembling into a pseudocapsid and preferably having an antigenic determinant in common with the wild type antigen. The capsid antigen can be a hybrid of polyomavirus major capsid antigens, for example a hybrid of human BK virus major capsid antigen and mouse polyoma virus VP1.
By the exogenous material being xe2x80x9cassociated withxe2x80x9d the pseudocapsid, we mean that the material is protected thereby. For example, exogenous DNA will be protected from degradation by DNases such as DNaseI, and exogenous protein will be protected from proteases. The exogenous material can be enclosed within an empty pseudocapsid or otherwise wrapped up with the capsid antigen.
The inventors have demonstrated that a simplified carrier consisting of a pseudocapsid involving only the major papovaviral capsid antigen can package and protect exogenous DNA, and transfer it into cells in a manner that allows for functional expression. A well-characterised gene, an efficiently transforming mutant form of the mouse polyoma virus middle T-antigen (MT), designated dl8MT (Griffin and Maddock, 1979), was chosen to show the worth of this system. The dl8-derived antigen possesses distinct physical and biological properties, which allow it to be easily assayed and distinguished from any wild type viral gene product that might have inadvertently contaminated recipient cells.
Papovaviruses have a broad host range and permissive cells include mammalian cells, such as rodent and human cells (Ponten, 1971). The pseudocapsids of the invention share this broad host range and can therefore be utilised in the transfer of exogenous material to a wide variety of cells.
The inventors have demonstrated that the pseudocapsids can successfully transfer exogenous genetic material in human cells.
Preferred host cells include mammalian cells, in particular rodents such as mice and rats, and human cells. Especially preferred human cells for in vivo gene transfer in accordance with the invention are lung cells.
Physical, non-viral gene transfer methods such as chemical and mechanical techniques and membrane fusion-mediated transfer via liposomes have been recently reviewed and assessed (Morgan and Anderson, 1993). The pseudocapsid (in the preferred embodiment involving genetic material) approach of the present invention resembles these procedures in that only the DNA intended for study, and no unwanted viral information, is introduced into cells. Moreover, the efficiency of functional gene transfer achieved by the method of the invention has been found to be between fifty and a hundred times greater than that achieved using conventional transfection methods.
The present data show that, in contrast to the calcium phosphatexe2x80x94or liposome-mediated route, long-term stable gene transfer and expression can be accomplished using pseudocapsids. Finally, it should be noted that whereas greater quantities of exogenous DNA are introduced into recipient rodent cells by the calcium phosphate technique, the latter route produces greater heterogencity in the delivered DNA than observed with the pseudocapsid approach, and is no more effective in terms of protein expression. The pseudocapsid approach is also advantageous in that it can be manipulated in a manner not applicable to chemical and physical transfer routes, making it of considerable value in future work including studies applied to gene therapy. The ease with which pseudocapsids can be produced and their efficient delivery of exogenous material, including the delivery of DNA to the cell nucleus, makes the present papovavirus pseudocapsid method of delivery especially attractive for use in therapy, especially gene therapy and antibody or drug targeting. A particular advantage of the pseudocapsid method of transfer is that it can deliver functional exogenous material to the nucleus of a cell.
In a further aspect the invention provides a pseudocapsid formed from papovavirus major capsid antigen and excluding minor capsid antigens, which pseudocapsid has exogenous material associated therewith, but excluding pseudocapsids associated with exogenous genetic material when the pseudocapsid is formed from only the mouse polyoma virus major capsid antigen, VP1. However, the invention does include pseudocapsids formed from mouse polyoma virus VP1 antigen if the exogenous genetic material and other exogenous material such as protein is associated with a pseudocapsid formed from only the mouse polyoma virus VP1 antigen.
In another aspect the invention provides a method of making a pseudocapsid comprising providing an empty pseudocapsid formed from papovavirus major capsid antigen and excluding minor capsid antigens; providing exogenous material; and mixing the empty pseudocapsid and exogenous material whereby the exogenous material becomes associated with the empty pseudocapsid. However, the invention does not include a method of making a pseudocapsid in which exogenous genetic material on its own is associated with an empty pseudocapsid formed from only the mouse polyoma virus major capsid antigen, VP1.
It should be noted that the invention does include a method of making a pseudocapsid in which both exogenous genetic material and other exogenous material such as protein is associated with a pseudocapsid formed from only the mouse polyoma virus VP1 antigen.
The term xe2x80x9cexogenous materialxe2x80x9d as used herein means material other than wild type papovavirus nucleic acid. Preferably, the material is genetic material, for example DNA.
Preferably, the exogenous material is derived from an eukaryotic source, in particular from a mammal such as a rodent or a human. By material xe2x80x9cderived fromxe2x80x9d we include material actually derived from a eukaryotic source, or obtained using eukaryotic material (for example cDNA obtained using eukaryotic mRNA) or synthesised to correspond to eukaryotic material (for example a chemically synthesised or recombinant copy of a eukaryotic cDNA, or an antisense oligonucleotide to eukaryotic genetic material).
Alternatively or additionally, the exogenous material may be a protein or other polypeptide, or any pharmacologically active compound, especially one which acts in or on the nucleus of cells.
In a preferred embodiment the exogenous material comprises a complex of two or more substances such as biological macromolecules. For example, if the sequence of the site to which DNA is to be targeted is known, this can be incorporated into the exogenous DNA together with the gene of interest, to promote homologous recombination at that site, using either double-stranded or single-stranded DNAs 20-200 bases long for this purpose. To promote the recombination event, single stranded recombinase binding proteins, such as the Escherichia coli RecA (B. J. Rao and C. M. Radding, Proc. Natl. Acad. Sci. U.S.A 91, 6161-6165, 1994) or its counterpart from S. Cerevisiae, Rad51, (A. Shinohara, H. Ogawa and T. Ogawa. Cell 69, 457 470, 1992) can be attached to DNA. Such a method can be used to greatly increase the efficiency and specificity of DNA integration into the host cell genome.
To demonstrate the effectiveness of the above method, for example, the dl8MT transforming gene of polyoma virus, to which a 20-200 long segment of mouse DNA has been ligated, with or without attached recombinase (or its active polypeptide site) can be incorporated into pseudocapsids and the ability of this carrier to produce dense foci, a marker of cellular transformation, monitored in vitro. For the mouse DNA sequence the results of D. M. Ding, M. D. Jones, A. Leigh-Brown and B. E. Griffin. EMBO. J. 1, 461-466, 1982; C. H. Streuli, N. S. Krauzewicz and B. E. Griffin, J. Virol. 64, 3570-3580 (1990) can be used. The comparative numbers of dense foci obtained are a marker of efficiency of the system and the effect produced by adding, for example, a recombinase protein. Sequence data obtained from chromosomal DNA from foci confirms the efficiency and specificity of the targeting. The present invention thus provides a method for specific targeting of exogenous DNA into a host chromosome.
A particularly preferred exogenous material for association with pseudocapsids of the invention comprises peptide-nucleic-acids (PNAs).
Peptide nucleic acids (PNAs) are single chain compounds consisting of a polyamide backbone having purine or pyrimidine bases attached to side chains, to form a base sequence superficially similar to that of a single chain nucleic-acid. Their structure, composition and synthesis is described by Nielsen, et al (1991), Science, 254, 1497-5001; Egholm et al (1992), American Chem. Soc, 114; and Cherny et al (1993), Proc. Natl. Acad. Sci. USA, 90, 1667-70.
PNAs are preferred exogenous material because they have properties that make them useful in therapy of diseases or to modify the structure of the genome of living cells by affecting gene expression.
In living cells, by binding one strand of complementary DNA in a duplex, and thereby displacing a portion of the other strand to form a D loop structure, PNAs can inhibit transcription of a gene, or cause DNA breakage as a consequence of the cellular enzymatic machinery attempting to repair the damage. Such breakage is a focus for an increased level of recombination with any homologous DNA strands present in the same pseudocapsid or delivered separately in another pseudocapsid. Such breakage can also kill the organism to which the DNA belongs. The organism either is the cell itself, or a protozoan parasite or a virus, etc. Since it is possible to synthesise unique PNA moieties having sequence specificities specific for pathogens such as viruses, including those like the retroviruses which may integrate their DNA into the genome of the host, PNAs can be used to selectively attack the target pathogen without any deleterious effect on the host genome.
Particularly preferred proteins or polypeptides are antibodies, recombinant antibodies, or fragments thereof.
The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact recognised by early protease digestion experiments. Further confirmation was found by xe2x80x9chumanisationxe2x80x9d of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, (1038), single-chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al, (1989) Nature 341, p44). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter and Milstein (1991) Nature 349, 293-299.
By xe2x80x9cScFv moleculesxe2x80x9d we mean molecules wherein the VH and VL partner domains are linked via a flexible oligopeptide.
The advantage of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
Whole antibodies, and F(abxe2x80x2)2 fragments are xe2x80x9cbivalentxe2x80x9d. By xe2x80x9cbivalentxe2x80x9d we mean that the said antibodies and F(abxe2x80x2)2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site.
In a further aspect the invention provides a pharmaceutical composition comprising a pseudocapsid formed from a papovavirus major capsid antigen and excluding minor capsid antigens, which pseudocapsid has a pharmacologically active compound associated therewith; the pseudocapsid being provided together with a pharmacologically acceptable carrier.
Preferably, the pharmacologically active compound is a chemotherapeutic agent.
Chemotherapeutic agents are used to eliminate cancerous tissue. Recently, agents in the form of proteins and other biological products including molecules of the immune system such as tumour necrosis factor, interleukins, interferons and monoclonal antibodies have been used for chemotherapy.
As discussed by Jain, R. K. (Scientific American July 1994: 42-49), to eradicate tumours chemotherapeutic agents must disperse throughout the growths in sufficiently high concentrations. However, it has been found that tumours are resistant to such dispersal. Conventional chemotherapeutic agents are small (molecular weight lower than 2000 daltons) and when administered to a mammal, leave the blood vessels and migrate through normal tissue mainly by diffusion. However, the above biological products are larger molecules (molecular weight above 5000 daltons) and travel mainly by convection, that is, transport in a stream of flowing fluid.
Convectionxe2x80x94dependent agents are inhibited from dispersing through the tumour because the uniform pressure in the tumour""s interior prevents convection from operating. This may explain why such agents display disappointing tumour eradication results in vivo.
Both large and small chemotherapeutic agents are susceptible to degradation before they reach their target cancer cells. Repeated dosage is therefore required to maintain the sufficiently high concentrations needed for tumour eradication. This is expensive and may produce an immune response that degrades the agent before it has the chance to work.
Other tumour barriers which reduce the effectiveness of chemotherapeutic agents include the lack of oxygen in tumours which may cause them to secrete large amounts of lactic acid. Many agents are broken down or fail to work in an acidic environment.
A current strategy for overcoming the above problem is to link an enzyme with an antibody specific for a tumour cell antigen to form an xe2x80x9cabzymexe2x80x9d. The enzyme is capable of converting a prodrug into an active form capable of killing a tumour cell. Large amounts of the abzyme are injected in the blood stream so that it can accumulate in the tumour. The prodrug is injected once the abzyme has cleared from normal tissues. As the prodrug is small it can diffuse into the tumour and be activated by the abzyme to kill cancer cells.
However, this strategy is not wholly effective because the prodrug can be degraded quickly and can seep back into vessels and be cleared away from tumours just as easily as it can diffuse out of blood vessels.
The pseudocapsid approach for delivering chemotherapeutic agents is advantageous because it protects the agents from degradation, during transport to target cancer cells.
Preferably, the exogenous material comprises all or a coding part of at least one gene. Advantageously, expression of the gene in the host cell has a therapeutic effect on the cell and/or a multi-cellular organism comprising that cell. Genes of particular interest are given in the following table:
This list indicates the principal current targets for gene therapy. Many of the diseases listed can be caused by defects in more than one gene; the gene defect listed is the defect targeted by current research.
If the exogenous material is protein it is protected from degradation by a protease. If the exogenous material is a pharmacologically active compound, it is protected from degradation caused by the surrounding environment.
The protection of exogenous material by the pseudocapsid from degradation can be determined readily. For example, to establish the levels of protection for DNA, the pseudocapsids can be packed with a radioactively labelled fragment, the buffer adjusted to 10 mM MgCl2 and then exposed for 20 min at 37xc2x0 C. to DNase I (100 xcexcg/ml). The DNase treated sample is then fractionated on a 10-40% sucrose gradient made in buffer A (see below) lacking glycerol to separate protected from digested DNA. The refractive index and radioactivity of collected fractions is measured. Most radioactivity is unincorporated DNA and is found on the top of gradient. Radioactive peaks banding within gradients corresponding to pseudocapsids (as shown by electron microscopy) indicate that the labelled DNA and pseudocapsids are associated and affording protection to the DNA. The amount of protected DNA typically represents 2-5% of total DNA measured.
Exogenous material may be packaged, that is, incorporated into an empty pseudocapsid. By xe2x80x9cemptyxe2x80x9d we mean that the pseudocapsid does not contain any papovavirus genetic material. It may of course contain other material, such as protein.
The attachment of protected exogenous material to the pseudocapsid, for example by coating with charged molecules, is a preferred method of associating the material with the empty pseudocapsid because it enables larger amounts of material to be transferred.
For example, larger fragments of exogenous genetic material can be packaged using an in vitro method such as that described below, and then further protected by the addition of charged molecules (such as poly-L-lysine or basic histones), followed by incubation for 30 mins. at 37xc2x0 C.
Binding charged molecules causes exogenous genetic material such as DNA to alter its"" conformation which affects the amount of genetic material that can be packaged into empty pseudocapsids.
The pseudocapsids used may be provided in the form of an icosahedron, like the wild type papovavirus capsid, or larger spherical or filamentous shapes which are advantageous because they permit packaging of larger nucleic acid fragments. The term xe2x80x9cpseudocapsidxe2x80x9d is thus not limited to particles which closely resemble wild type papovavirus capsids.
The transfer of larger amounts of exogenous material may be desirable. For example transfer of larger fragments of genetic material is advantageous because there appears to be a size constraint on the amount of genetic material that can be packaged and transferred to host cells, using the empty icosahedral VP1 pseudocapsids, for example. The wild type viral genome of papovaviruses is 5.2 kbp and, using the passive DNA packaging procedures of Barr et al. (1979) or Slilaty et al. (1982), the amount of DNA that can be packaged is around 3 kbp. Whereas this size would be adequate for expression of many mammalian cDNAs, or, for example, oligonucleotides to be used for antisense xe2x80x9cknockoutxe2x80x9d experiments (ie where a gene function is deleted), it would preclude packaging of a number of mammalian genes in their entirety.
Recombinant baculoviruses carrying genes for papovaviruses major capsid antigen, such as the mouse polyoma virus major capsid antigen VP1, have been constructed and the antigens can be expressed in insect cells as described by Forstovxc3xa1 et al. 1993. In this method insect cells are infected with a recombinant baculovirus expressing VP1, harvested 72 hours post infection, resuspended in buffer A (50 mM NaCl, 10 mM Tris-HCl, pH 7.4. 0.01 mM CaCl2, 0.01% Triton X-100) and disrupted by sonication.
Cell debris is removed by low speed centrifugation and VP1 pseudocapsids purified on a CsCl density gradient. Peak fractions of pseudocapsids are subjected to centrifugation through a sucrose gradient (10-40%) and peak fractions (monitored by immunoblotting and electron microscopy) are then concentrated by CsCl gradient centrifugation. After each centrifugation the pseudocapsids are dialysed into buffer A.
In a preferred method, Sf9 insect cells are incubated at a multiplicity of infection of 10-20 with a recombinant baculovirus expressing VP1, harvested 72 hrs postinfection, resuspended in buffer A (150 mM NaCl, 10 micromolar CaCl2, 10 mM Tris-HCl, pH 7.6, 5% glycerol, 0.01%, Triton X-100) and disrupted by sonication. Cell debris is removed by low speed centrifugation at 8000xc3x97g. The empty VP1 pseudocapsids in the supernatant are concentrated by pelleting through a sucrose cushion and then by banding on a CsCl gradient. The fractions containing purified empty pseudocapsids, as revealed by refraction index measurements or electron microscopy, are dialysed into buffer A.
Using the above methods the yield of empty VP1 pseudocapsids is at least ten times greater than that achieved when the same procedure is carried out by co-infection of insect cells with recombinant baculoviruses expressing VP1, and the minor capsid antigens VP2 and VP3. Furthermore, empty VP1 pseudocapsids are just as efficient as VP1, VP2 and VP3 capsids at packaging exogenous genetic material.
Empty pseudocapsids formed from other papovavirus major capsid antigens can be produced using similar methods.
Protecting the exogenous material with an empty pseudocapsid is conveniently achieved for exogenous DNA, for example, by mixing 1 xcexcg DNA with 10-60 xcexcg of empty pseudocapsids and incubating the mixture in 150 mM NaCl, 0.01 mM CaCl2 10 mM Tris, at pH 7.4. 0.01% TX-100, for 10 minutes at 37xc2x0 C. The mixture is then subjected to osmotic shock by quick dilution with water to a final concentration of 35 mM NaCl2 with incubation for an additional 20 minutes. The pseudocapsids incorporating exogenous DNA can then be used in the exogenous material transfer method of the invention.
Exogenous material, in the form of protein and polypeptides such as antibodies and fragments thereof, and pharmacologically active compounds, can be packaged inside an empty pseudocapsid by a similar procedure in which the DNA is substituted in the above method by the exogenous material to be packaged.
Alternatively, exogenous material is associated with empty pseudocapsids by dissociating the empty pseudocapsid structure and reassembling it in the presence of the material.
An advantage of the pseudocapsids of the invention is that they are easier to produce than capsids containing both major and minor capsid antigens. This is because it is difficult to control the proportions of the various capsid antigens in the assembled capsid. Consequently, the properties of the capsids produced will vary and this unpredictability is undesirable.
A further advantage of the present pseudocapsids for in vivo exogenous material transfer over known capsids is that they are less likely to evoke a strong immune response than capsids because they only contain a single foreign antigen, the major capsid antigen.
Treatment of host cell with pseudocapsids in accordance with the invention can be carried out by washing the cells in serum free medium and incubating them with pseudocapsids diluted to 1 ml/106 cells with tissue culture medium. Following 90 mins incubation, medium containing serum is added to the culture. For example, 15 xcexcg of pseudocapsids containing an agarose gel-purified fragment (2.5 xcexcg) of the cellular gene p43 (Koch et al., 1990) under the control of the cytomegalovirus immediate early promoter can be used to treat human embryo lung (HEL) fibroblast cells, using 2xc3x97102 cells. The cells are then incubated at 37xc2x0 C. for 72 hours post treatment.
To test for functioning of the p43 fragment, cells are harvested by washing with phosphate buffered saline (PBS), and lysing in Laemmli sample buffer (Laemmli, 1970). Proteins are fractionated on 10% SDS-PAGE, immunoblotted and probed initially with a p43 specific monoclonal antibody, then anti-xcex2 tubulin antibody (Sigma). Immune complexes are detected with an HRP conjugated secondary antibody (Dako plc) and chemiluminescent ECL reagent (Amersham plc).